The berry firmness was then determined . In the 2015 season, three grape clusters of uniform size and at the same phenological stage were identified in each plot, and two berries were collected randomly from each bunch at each sampling time. The berry skins were removed, frozen, kept at −80◦C, and transferred to the Department of Viticulture and Enology of the University of California, Davis, CA, United States, for further analyses. The skins were then placed in liquid nitrogen and ground using a TissueLyser II . RNA was extracted using 0.5 g of ground tissue based on the protocol described by Blanco-Ulate et al. . RNA concentration and purity were determined using a NanoDrop 2000c Spectrophotometer , and RNA integrity was checked by electrophoresis on 1.5% agarose gel. Reverse transcription was performed using 1 µg of RNA and M-MLV Reverse Transcriptase according to the manufacturer’s instructions. qRT-PCRs were performed using the SYBRR Green PCR Master Mix kit . The PCR program consisted of 70◦C for 10 min, 36 cycles at 42◦C for 2 s, and 37◦C for 50 min. VvActin was used as the reference gene and processed in parallel with the genes of interest. Gene sequences used for primer design were obtained from the GenBank of the National Center for Biotechnology Information using Primer-BLAST software . The relative levels of target gene expression were calculated using the formula 2 . The linearized values correspond to the relative gene expression within a given sample and are comparable across genes. Four biological replicates of S-ABA treated and control grape berries were used to obtain the relative gene expression data.Application of abscisic acid increased the total anthocyanin concentration in berry skins of the seedless grapeSelection 21 during the 2013 and 2014 seasons, regardless of the S-ABA concentration and time of application .

However, berries that received 400 mg/L of S-ABA at 7 and 21 DAV had significantly higher, almost two to three times more, 25 liter pot anthocyanin concentrations than other treatments. According to the CIRG, berries from control treatments had a green to a yellow color in both seasons . In 2013, berries treated with one or two applications of 200 mg/L S-ABA or one application of 400 mg/L S-ABA at 7 DAV, and those in the 2014 season that were treated with one application of 200 mg/L S-ABA developed a pink color . Remarkably, berries of the 2013 season treated with two applications of 400 mg/L S-ABA and berries of the 2014 season treated with one or two 400 mg/L S-ABA applications, developed a stronger red color . For both the 2013 and 2014 seasons, color coverage was lowest in control grapes and highest in grapes treated with two applications of 400 mg/L S-ABA. Increase in total polyphenols was evident in grapes subjected to two 400 mg/L S-ABA applications during the 2013 and 2014 seasons. These berries also presented the lowest mean berry firmness . Importantly, the increased softening due to S-ABA application did not result in higher frequency of cracked berries in any of the studied seasons. Qualitative assessment of berry cracking was performed visually. Further analyses of the effect of 400 mg/L S-ABA treatments on CIRG, total and individual anthocyanins concentrations, and gene expression of transcription factors and biosynthetic enzymes were performed with grape berries collected from the 2015 trial. Measurements of CIRG confirmed previous results obtained during the 2013 and 2014 seasons, at the time of harvest , grapes treated with two S-ABA applications had the highest CIRG values . Grape bunches from the control treatment presented pink berries , whereas those treated with one or two applications of S-ABA had red berries . As determined in the previous seasons, berries treated with 400 mg/L S-ABA also presented higher total anthocyanin content than the control at 14 and 28 DAV . At 28 DAV, grapes treated with one or two applications of 400 mg/L S-ABA presented total anthocyanin concentrations almost three times higher than the control.

Even 3 weeks after the first application , berries treated with only one S-ABA application showed a total anthocyanin content similar to those treated with two S-ABA applications. Nonetheless, the second application of 400 mg/L S-ABA significantly affected the total anthocyanin accumulation at the time of harvest . With the exception of petunidin- 3-glucoside, S-ABA application significantly improved the concentrations of all the measured anthocyanins. Cyanidin-3- glucoside and peonidin-3-glucoside levels increased at 14 DAV, 1 week after the first S-ABA application. The second S-ABA application stimulated the accumulation of the anthocyanin delphinidin-3-glucoside at 28 DAV, yielding differences relative to both the control and to the samples treated with only one S-ABA application. At 28 DAV, the concentrations of peonidin-3-glucoside and malvidin-3-glucoside increased after exogenous S-ABA application but were not further increased by the second application. At the time of harvest , peonidin-3-glucoside and cyanidin-3-glucoside were the dominant pigments present after all treatments. Delphinidin- 3-glucoside, cyanidin-3-glucoside, and peonidin-3-glucoside presented higher accumulation following the second application of 400 mg/L S-ABA, but the number of applications did not affect the accumulation of malvidin-3-glucoside. As presented in Figure 4, treatment with 400 mg/L S-ABA significantly increased the expression of the transcription factors VvMYBA1 and VvMYBA2 and the expression of the biosynthetic genes CHI, F3H, DFR, and UFGT 1 week after the first application . Three weeks after the first S-ABA application , expression of CHI, F3H, and DFR genes remained high, but this was not observed for the transcription factors VvMYBA1 and VvMYBA2 or the UFGT gene. Four weeks after the first S-ABA application , no significant differences were observed in the expression of genes or transcription factors between berries that received one S-ABA application and those that received the control treatment.

The two applications of 400 mg/L S-ABA induced expression of the genes CHI, F3H, DFR, and UFGT and the transcription factors VvMYBA1 and VvMYBA2 at 14 and 28 DAV . F3H expression was the most affected by S-ABA application, displaying higher levels than the control until the final stages of berry maturation at 35 DAV,whereas the remaining genes presented no differences from the control at harvest. Overall, the gene expression results indicate that a second S-ABA application contributed to the maintenance of the expression of the transcription factors VvMYBA1 and VvMYBA2 and the genes F3H and UFGT at higher levels than in the control for an extended period of time.Exogenous application of S-ABA improves the color of table grapes by stimulating the anthocyanin synthesis and accumulation in the grape skin . Our results are similar to those reported previously for “Flame Seedless” grapes, in which applications of 300 mg/L ABA during or after véraison were more effective at increasing anthocyanin concentrations than application before véraison . In ‘Crimson Seedless’ grapes, anthocyanin concentrations increased with application of 400 mg/L S-ABA at 17 DAV, but the response varied widely between seasons depending on S-ABA dosage and time of application . Exogenous S-ABA application is thought to simulate plant stress responses and accelerate ripening processes . High ABA concentrations are believed to be perceived by grapevines as a drought stress signal . Subsequently, water stress leads to changes in grape secondary metabolism, significantly increasing flavonoid levels and, especially, anthocyanin biosynthesis . In our study, in addition to increasing anthocyanin concentration, exogenous S-ABA improved both berry color intensity and uniformity. This is important because the visual assessment of berry color characteristics determine, in part, the commercial value of table grapes . Grape clusters with more intense and uniform berry color have higher consumer acceptance. Improved color characteristics such as increased color coverage of grape berries, and uniformity of berry color within a cluster, were also observed in “Benitaka” and “Rubi” table grapes following application of 400 mg/L S-ABA at 7 DAV and 15 days before harvest . In our experiments, berries from the seedless grape Selection 21 treated with one or two applications of 400 mg/L S-ABA presented higher CIRG values than the controls. Besides, 25 liter plant pot improving cluster color and attractiveness, exogenous S-ABA can potentially decrease the time to harvest, a feature that is very advantageous for grape commercialization . The observed increase in anthocyanin concentration resulting from S-ABA application does not necessarily result in an increase in total polyphenol content; polyphenols include phenolic acids, stilbenes, coumarins, tannins, and flavonoids , as reported in “Alachua” muscadine grapes and in “Isabel” grapes . Environmental factors such as temperature, rainfall, and altitude could also influence berry polyphenol concentrations. In addition, the berry ripening stage is directly correlated to the concentration and proportion of several polyphenols that impact the organoleptic properties, nutritional value, and antioxidant capacities of the grapes . S-ABA application can negatively affect berry firmness, an important characteristic for the successful postharvest handling of grapes for the fresh fruit market because it influences transportability and shelf life . ABA application is known to cause loosening and decreased rigidity of the cell wall, resulting in fruit softening and a higher probability of berry cracking .

Treatment of grapes with exogenous S-ABA can result in changes in the regulation of proline-rich cell wall proteins and in the induction of cell wall degrading genes, such as polygalacturonases that promote pectin solubilization and depolymerization . The effect of S-ABA on berry firmness was also observed in “Flame Seedless” grapes, where it caused softening similar to that caused by ethephon application , as well as in ‘Crimson Seedless’ and “Red Globe” grapes . Therefore, it is still necessary to evaluate if the benefits of applying exogenous ABA to improve berry color can outweigh a potential reduction in the shelf life of treated grapes.Multiple applications of exogenous ABA can promote anthocyanin accumulation for longer periods of time . It is possible that more than one ABA application could induce a milder response at later grape phenological stages or that the effects of a second application could take more time to be evident. In this study, the second application of 400 mg/L S-ABA significantly increased the total anthocyanin content at the time of harvest, which supported the latter hypothesis and confirmed that two S-ABA applications had a more pronounced effect than only one application. The higher total anthocyanin concentration observed with S-ABA application appeared to result from a transient effect of S-ABA, because the anthocyanin concentration of grapes that received only one S-ABA application remained essentially constant between 28 and 35 DAV. It may, therefore, be inferred that the action of S-ABA decreases over time and that its levels increase with a second application, allowing maintenance of its activity. Three applications of 400 mg/L S-ABA at 1-week intervals prior to véraison resulted in an earlier accumulation of anthocyanin in “Cabernet Sauvignon” grapes, but no differences in anthocyanin concentration at harvest were observed in grapes that received different treatments . The increase in endogenous ABA concentration in grape berries occurs at the beginning of véraison and extends until the establishment of maturation when endogenous ABA concentrations peak. ABA concentration then decreases until harvest, the period over which the decrease occurs ranges from 13 to 20 days depending on the cultivar . Application of exogenous S-ABA close to véraison, when ABA naturally reaches its highest concentration in berries, was shown to be more effective in increasing anthocyanin accumulation than application at other times . S-ABA application significantly increased endogenous ABA levels 7 days after application in “Carménère” grapes, 40 days later, the ABA levels in the treated berries remained higher than those from control .dihydroxylated precursors of red anthocyanins in grape skin, whereas delphinidin, petunidin, and malvidin are trihydroxylated precursors of blue and purple anthocyanins . Accumulation of individual anthocyanins in grapes may be induced by S-ABA application and varies with the cultivar. In “Noble” and “Alachua” muscadine grapes, application of S-ABA during véraison and again at 8 DAV for “Noble” or again at 13 DAV for “Alachua” resulted in higher levels of accumulation of all evaluated anthocyanins in “Noble” grapes but not in “Alachua” grapes, which only presented higher accumulation of peonidin-3-diglucoside compared to the control . Changes in the proportions of individual anthocyanins resulting from S-ABA application were also observed in “Cabernet Sauvignon” grapes, both in berries and in wine . In “Isabel” grapes, application of S-ABA increased the accumulation of individual anthocyanins both in must and in processed whole juice .